Recombinant Vibrio vulnificus 30S ribosomal protein S21 (rpsU)

What is the role of S21 protein in the 30S ribosomal subunit of V. vulnificus?

S21 is one of the 21 proteins (S1-S21) that constitute the small 30S ribosomal subunit in prokaryotes including Vibrio vulnificus. It plays a critical role in ribosome assembly and function, particularly in the later stages of 30S subunit assembly. Unlike some other ribosomal proteins, S21 has been noted to have proline at its N-terminus in its native form, which can affect its incorporation into the ribosomal structure. In recombinant expression systems, this proline is sometimes replaced with alanine to facilitate protein production and purification processes .

How does S21 compare structurally and functionally across different bacterial species?

While the search results don't provide direct comparative data for S21 across species, ribosomal proteins generally show conservation in their core functions while exhibiting species-specific variations. In the case of S21 specifically, one notable feature in V. vulnificus and other bacteria is the presence of an N-terminal proline residue. During recombinant expression studies, researchers have noted that this proline is sometimes replaced with alanine to facilitate expression and purification, which suggests that this residue has structural significance but may not be absolutely essential for basic function in reconstitution experiments .

What is the most efficient method for recombinant expression of V. vulnificus S21 protein?

The most efficient method documented for recombinant expression of V. vulnificus S21 protein involves the small ubiquitin-related modifier (SUMO) fusion method. This approach begins with expressing S21 as a His-tagged SUMO fusion protein in E. coli expression systems. The procedure involves:

• Cloning the S21 coding sequence directly downstream from the SUMO coding end

• Expressing the fusion protein in E. coli

• Purifying using affinity chromatography (typically Ni-NTA)

• Cleaving the His-tagged SUMO component using Ulp1 protease (Ulp1p), which specifically recognizes and cleaves after the SUMO coding end

• Final purification to obtain the native S21 protein

For S21 specifically, as it naturally has a proline at the N-terminus, researchers often replace this proline with alanine to facilitate the SUMO fusion method .

What purification protocols yield the highest purity and activity of recombinant S21?

The highest purity and activity of recombinant S21 is achieved through a multi-step purification protocol:

• Initial purification using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography to capture His-tagged SUMO-S21 fusion protein

• Cleavage with Ulp1 protease to remove the SUMO tag

• Secondary chromatography steps to separate the cleaved S21 from the His-SUMO tag

• Quality control through SDS-PAGE analysis to confirm size and purity

This protocol has been shown to yield functional S21 protein that, when combined with other purified ribosomal proteins and 16S rRNA, can form active 30S subunits capable of participating in poly(U)-directed polyphenylalanine synthesis and full-length protein synthesis in the PURE system .

How can researchers verify the structural integrity of purified recombinant S21?

Researchers can verify the structural integrity of purified recombinant S21 through several complementary approaches:

• SDS-PAGE Analysis: To confirm the correct molecular weight and initial purity

• Western Blot Analysis: Using anti-S21 antibodies for specific identification

• Functional Reconstitution Assays: Incorporating S21 into 30S subunit reconstitution experiments and measuring the resulting ribosomal activity

• Sucrose Density Gradient (SDG) Analysis: To confirm proper integration of S21 into the 30S subunit structure

• Mass Spectrometry: For precise mass determination and identification of post-translational modifications

The most definitive verification comes from functional assays, where the purified S21 is used alongside other ribosomal components to reconstitute 30S subunits. If these reconstituted subunits demonstrate activity in translation assays such as poly(U)-directed polyphenylalanine synthesis or full protein synthesis in the PURE system, this provides strong evidence that the purified S21 retains its native structural properties .

What is the optimal protocol for incorporating S21 into reconstituted 30S subunits?

The optimal protocol for incorporating S21 into reconstituted 30S subunits involves two main approaches, depending on the experimental conditions desired:

Under High-Salt Conditions (Conventional Method):

• Prepare individually purified ribosomal proteins S2-S21 using the SUMO fusion method

• Isolate 16S rRNA from native 30S subunits

• Form reconstitution intermediate (RI) by incubating 16S rRNA with ribosomal proteins at 40°C for 20 minutes in buffer containing high salt (330 mM KCl, 20 mM MgCl₂)

• Heat activation of the RI at 42°C to achieve final 30S structure

• Purify reconstituted 30S subunits using sucrose density gradient centrifugation

Under Physiological Conditions (with Biogenesis Factors):

• Prepare individually purified ribosomal proteins S2-S21

• Isolate 16S rRNA

• Include specific biogenesis factors, particularly GTPases Era and YjeQ

• Incubate at 37°C in buffers with near-physiological salt concentrations (150 mM K⁺, 5 mM Mg²⁺)

• Purify reconstituted 30S subunits

The incorporation of S21 can be verified by SDS-PAGE analysis of the purified reconstituted 30S subunits .

How does the presence or absence of S21 affect the functional activity of reconstituted 30S subunits?

Fully reconstituted 30S subunits containing all proteins (S2-S21) typically display approximately 30% of the polyphenylalanine synthesis activity of native 30S subunits. This indicates that while S21 is part of the functional ribosome, other factors such as proper assembly kinetics and potentially post-translational modifications also contribute to full ribosomal activity.

Interestingly, research has shown that the addition of protein S1 (which is not always included in initial reconstitution experiments) to reconstituted 30S subunits containing S2-S21 increases their activity by more than twofold. This suggests potential cooperative interactions between ribosomal proteins, including S21, in achieving optimal translation activity .

What biogenesis factors are most critical when working with recombinant S21 in 30S subunit assembly?

When working with reconstituted 30S subunits containing recombinant proteins including S21, several biogenesis factors have been identified as critical for efficient assembly:

• GTPases Era and YjeQ: These have been shown to dramatically affect 30S subunit assembly under physiological conditions. They likely facilitate the proper positioning of ribosomal proteins and assist in the maturation of the 30S structure.

• RNA helicases: While specific details are not provided in the search results, these enzymes help resolve RNA secondary structures that might impede proper ribosome assembly.

The importance of these factors becomes particularly evident when attempting to reconstitute 30S subunits under physiological conditions (lower salt concentrations), which more closely resemble the cellular environment in which ribosomes naturally assemble. Under these conditions, the efficiency of reconstitution is significantly enhanced by the presence of these biogenesis factors.

This table summarizes the effect of biogenesis factors on 30S reconstitution:

The use of these factors represents an important step toward developing systems that can reconstitute ribosomes under conditions that more closely mimic the cellular environment .

How can recombinant S21 be used to study ribosome assembly pathways in V. vulnificus?

Recombinant S21 can be strategically employed to study ribosome assembly pathways in V. vulnificus through several sophisticated experimental approaches:

• Fluorescently Labeled S21: By introducing fluorescent tags to recombinant S21, researchers can track its incorporation into assembling ribosomes in real-time using techniques such as fluorescence resonance energy transfer (FRET).

• Assembly Kinetics Studies: Using purified recombinant S21 in time-course reconstitution experiments to determine the precise timing of S21 incorporation into the assembling 30S subunit.

• Mutational Analysis: Introducing specific mutations into recombinant S21 to identify critical residues for ribosome assembly and function.

• Cryo-EM Structural Studies: Using recombinant S21 in reconstitution experiments for structural analysis of assembly intermediates at different stages.

• Comparative Assembly Studies: Reconstituting 30S subunits under different conditions (high-salt vs. physiological conditions with biogenesis factors) to understand how the assembly pathway differs in more native-like environments.

The ability to reconstitute 30S subunits from individually purified components, including recombinant S21, allows researchers to manipulate and observe the assembly process in ways not possible with in vivo systems .

What insights can be gained from studying post-translational modifications of S21 in V. vulnificus?

Studying post-translational modifications (PTMs) of S21 in V. vulnificus can provide critical insights into ribosome function and regulation:

• Functional Impact Assessment: Comparing the activity of reconstituted 30S subunits containing native S21 (with PTMs) versus recombinant S21 (lacking PTMs) can reveal the functional significance of these modifications. Research has shown that reconstituted 30S subunits containing recombinant proteins exhibit approximately 30% of the activity of native 30S subunits, suggesting that PTMs may contribute to optimal ribosomal function.

• N-terminal Acetylation: The current recombinant expression system using SUMO fusion does not introduce N-terminal acetylation, which is a common PTM in native ribosomal proteins. This provides an opportunity to study the specific contribution of this modification to S21 function.

• Proline Modification: Native S21 contains an N-terminal proline, which is often replaced with alanine in recombinant expression systems. This substitution provides a model to study how proline-specific modifications affect ribosome assembly and function.

• Regulatory Mechanisms: PTMs may play roles in regulating ribosome assembly or activity in response to changing cellular conditions, potentially linking translation to broader cellular signaling networks.

Understanding these modifications could provide insights into evolutionary adaptations of the V. vulnificus translation machinery and potentially reveal novel regulatory mechanisms specific to this pathogenic organism .

How does rpsU gene regulation compare to other ribosomal protein genes in V. vulnificus?

While the search results don't provide direct information about rpsU (S21) gene regulation in V. vulnificus, we can make some inferences and comparisons based on the information about rpoS regulation and general principles of ribosomal protein gene regulation:

• Potential Feedback Mechanisms: Like other ribosomal protein genes, rpsU expression might be regulated through feedback mechanisms where excess free S21 protein binds to its own mRNA to prevent further translation.

• Growth Phase-Dependent Regulation: Given that rpoS in V. vulnificus shows complex regulation with two distinct promoters (proximal and distal) that are active under different conditions, rpsU might similarly show differential expression patterns during various growth phases.

• cAMP-CRP Regulation: The search results indicate that in V. vulnificus, rpoS expression is repressed by the cAMP-CRP complex. It would be valuable to investigate whether rpsU and other ribosomal protein genes are regulated by similar mechanisms, which would suggest coordinated regulation of stress response and translational machinery.

• Stress-Responsive Regulation: Since rpoS is a stress-response regulator in V. vulnificus, it's possible that it might in turn regulate rpsU expression under stress conditions, creating a regulatory network that coordinates stress response with translational capacity.

Comparative studies of rpsU regulation with other ribosomal protein genes could reveal whether V. vulnificus employs unique regulatory mechanisms for different components of its translational machinery, potentially reflecting adaptations to its specific ecological niche and pathogenic lifestyle .

What are the main challenges in achieving high-yield expression of recombinant V. vulnificus S21?

Researchers face several key challenges when attempting to achieve high-yield expression of recombinant V. vulnificus S21:

• N-terminal Proline Issue: S21 naturally contains proline at its N-terminus, which can complicate certain expression systems. Researchers often need to replace this proline with alanine when using the SUMO fusion method to facilitate proper expression and cleavage.

• Protein Solubility: Ribosomal proteins, including S21, tend to be highly basic and can form insoluble aggregates when expressed at high levels. Optimization of expression conditions (temperature, induction time, media composition) is often necessary.

• Proper Folding: Ensuring proper folding of S21 outside its natural ribosomal context can be challenging. The use of fusion partners like SUMO can help improve solubility and folding.

• Proteolytic Degradation: Small ribosomal proteins may be susceptible to proteolytic degradation during expression and purification. Protease inhibitors and optimized purification protocols can help mitigate this issue.

• Purification Efficiency: Achieving high purity of S21 after tag removal can be challenging. Multiple chromatography steps may be required to separate the cleaved protein from the tag and contaminants.

The SUMO fusion method has proven to be an effective approach to overcome many of these challenges, allowing for the successful expression and purification of functional recombinant S21 protein .

How can researchers troubleshoot low activity in reconstituted 30S subunits containing recombinant S21?

When facing low activity in reconstituted 30S subunits containing recombinant S21, researchers can implement the following troubleshooting strategies:

• Verify S21 Structural Integrity: Ensure recombinant S21 has the correct sequence and structural properties using mass spectrometry and circular dichroism.

• Add S1 Protein: Research has shown that adding S1 protein to reconstituted 30S subunits can increase their activity by more than twofold in polyphenylalanine synthesis assays.

• Include Biogenesis Factors: Incorporate GTPases Era and YjeQ, which have been demonstrated to dramatically improve 30S subunit assembly under physiological conditions.

• Optimize Reconstitution Conditions: Adjust salt concentrations, temperature, and incubation times during the reconstitution process. Consider testing both high-salt conditions (traditional method) and more physiological conditions.

• Examine Assembly Order: Ensure proper sequential incorporation of ribosomal proteins, as the assembly pathway can significantly impact final ribosomal activity.

• Heat Activation Step: Include a heat activation step (42°C) to promote proper maturation of the 30S structure, which has been shown to enhance activity.

• Check 16S rRNA Quality: The integrity and modifications of 16S rRNA significantly impact ribosomal activity. If using in vitro transcribed rRNA, consider using native rRNA which contains important modifications.

Implementation of these strategies has been shown to improve reconstituted 30S subunit activity from approximately 30% to up to 80% of native 30S subunit activity in polyphenylalanine synthesis assays .

What are the critical quality control parameters for assessing recombinant S21 before use in ribosome reconstitution experiments?

Before using recombinant S21 in ribosome reconstitution experiments, researchers should evaluate several critical quality control parameters:

• Purity Assessment:

  • SDS-PAGE analysis showing a single band at the expected molecular weight

  • Absence of contaminating proteins or degradation products

  • Purity level >95% as determined by densitometric analysis

• Identity Confirmation:

  • Mass spectrometry to confirm the exact molecular mass and sequence

  • Western blot using S21-specific antibodies if available

  • N-terminal sequencing to verify correct processing of the fusion protein

• Structural Integrity:

  • Circular dichroism (CD) spectroscopy to assess secondary structure elements

  • Thermal stability analysis to determine melting temperature

  • Native PAGE to evaluate oligomerization state

• Functional Testing:

  • Binding assays with 16S rRNA fragments

  • Preliminary small-scale reconstitution tests

  • RNA protection assays to verify interaction with target rRNA regions

• Storage Stability:

  • Evaluation of activity after freeze-thaw cycles

  • Testing of different buffer conditions for optimal stability

  • Quantification of aggregation propensity under storage conditions

The most definitive quality control test is to use the purified S21 in a complete 30S subunit reconstitution experiment followed by functional testing. If the reconstituted subunits show substantial activity in poly(U)-directed polyphenylalanine synthesis or full-length protein synthesis in the PURE system, this confirms that the recombinant S21 is functional .

How does ribosomal protein S21 contribute to stress response and virulence in V. vulnificus?

While the search results don't provide direct evidence about S21's specific role in V. vulnificus virulence, we can integrate available information to form a hypothesis:

As a component of the 30S ribosomal subunit, S21 likely plays an indirect but important role in stress response and virulence through its contribution to translation. In V. vulnificus, the global stress regulator RpoS has been shown to be crucial for stress resistance and virulence factor expression. For RpoS to exert its effects, functional ribosomes containing properly assembled 30S subunits (including S21) are essential to translate the stress-response proteins and virulence factors.

The integration between ribosomal function and stress response is further evidenced by the regulation of rpoS expression in V. vulnificus by the cAMP-CRP complex, suggesting a coordinated regulatory network linking central metabolism, stress response, and potentially ribosome biogenesis. Under stress conditions that V. vulnificus experiences during infection (e.g., host-derived stresses), the proper functioning of ribosomes becomes crucial for the pathogen to adapt and express virulence factors.

Further research specifically examining the role of S21 in translation of stress-response and virulence genes would be valuable to establish a more direct connection between this ribosomal protein and V. vulnificus pathogenesis .

Can recombinant S21 be used to develop novel antimicrobial strategies against V. vulnificus?

Recombinant S21 presents several potential avenues for developing novel antimicrobial strategies against V. vulnificus:

• Structure-Based Drug Design: Recombinant S21 can be used in structural studies to identify unique binding pockets or structural features that differ from human ribosomal proteins. These differences could be exploited to design selective inhibitors that target V. vulnificus ribosomes without affecting host ribosomes.

• Ribosome Assembly Inhibitors: Understanding the incorporation of S21 into the 30S subunit could lead to the development of compounds that specifically interfere with ribosome assembly in V. vulnificus. The reconstitution systems described in the research provide valuable platforms for screening such inhibitors.

• Decoy Strategies: Engineered variants of S21 that can compete with native S21 but render ribosomes non-functional could potentially be developed as antimicrobial agents.

• Epitope Mapping for Immunotherapeutics: If S21 contains surface-exposed regions on the assembled ribosome, recombinant S21 could be used to develop antibodies or immunotherapeutics that target V. vulnificus ribosomes.

• Combination Approaches: Targeting S21 function in conjunction with other antimicrobial strategies might enhance efficacy, particularly against drug-resistant strains.

These approaches could be particularly valuable given the increasing antibiotic resistance among bacterial pathogens and the life-threatening nature of V. vulnificus infections, which often require rapid and effective treatment .

How does temperature affect S21 incorporation into ribosomes and what are the implications for V. vulnificus environmental adaptation?

While the search results don't directly address temperature effects on S21 incorporation specifically, they do provide insights into temperature-dependent aspects of ribosome assembly that can inform our understanding of V. vulnificus environmental adaptation:

• Heat Activation in Reconstitution: In vitro reconstitution experiments typically include a heat activation step at 42°C to promote proper 30S subunit assembly. This suggests that temperature plays a critical role in ribosome maturation, potentially affecting S21 incorporation.

• Temperature-Dependent Assembly Kinetics: The research shows that reconstituted 30S subunits exhibit different properties depending on whether they undergo heat treatment. This implies that temperature shifts could influence the pathway and efficiency of ribosome assembly, including S21 incorporation.

• Environmental Adaptation Implications: V. vulnificus is found in marine and estuarine environments and can cause human infections, experiencing temperature fluctuations ranging from environmental water temperatures (~15-30°C) to human body temperature (37°C). The ability to efficiently incorporate S21 and other ribosomal proteins across this temperature range would be crucial for the pathogen's ability to adapt to different environments.

• Potential Temperature-Dependent Regulation: Temperature shifts experienced during host invasion might trigger changes in ribosome composition or assembly pathways as part of the adaptive response, potentially involving modified incorporation of S21.

This temperature dependency of ribosome assembly has significant implications for understanding how V. vulnificus adapts to environmental transitions, particularly the shift from marine environments to human hosts during infection. Further research specifically examining S21 incorporation at different temperatures would provide valuable insights into this aspect of V. vulnificus biology .